The search for improved chemotherapeutic agents with novel mechanisms of action is driven by several, important, unmet clinical needs. First and foremost, almost all anticancer drugs cause extreme secondary side effects. Moreover, solid tumors, which are subject to abnormally high interstitial pressure, tend to be resistant to drug penetration by simple diffusion. Since diffusion rates correlate closely with molecular weights, the utility of anticancer strategies that rely on high molecular weight species such as monoclonal antibodies, tumor necrosis factor, interleukins, interferons, and other macromolecules for tumor therapy may ultimately be limited. For that reason, the development of new chemotherapeutic agents and strategies based on small molecules that can be targeted selectively to diseased tissues continues to be an important undertaking. Research over the past several decades indicates that some of the best new lead compounds for cancer chemotherapy have emerged from a better understanding of natural compounds possessing antitumor or anticancer activity.
In 1975, the fungal metabolite 2-crotonyloxymethyl-(4R, 5R, 6R)-4,5,6-trihydroxy-2-cyclohexenone (COTC, 1a, Scheme 1) was found to display potent antitumor activity in vitro (Takeuchi, T.; Chimura, H.; Hamada, M.; Umezawa, H.; Yoshka, H.; Oguchi, N.; Takahashi, Y.; Matsuda, A. A Glyoxalase I Inhibitor of a New Structural Type Produced by Streptomyces. J. Antibiot. 1975, 28, 737-742). Besides that antitumor activity, it was noted that COTC reacted with reduced glutathione (GSH) to form a new product that inhibited glyoxalase I, a key enzyme in the detoxification of methylglyoxal. In 1975, Chimura et al. attributed the antitumor activity of 1a to its putative glutathione (GSH) adduct 2a, which was proposed to inhibit the enzyme glyoxalase I (GlxI) (Chimura, H.; Nakamura, H.; Takita, T.; Takeuchi, T.; Umezawa, M.; Kato, K.; Saito, S.; Tomisawa, T.; Iitaka, Y. The Structure of a Glyoxalase I Inhibitor and Its Chemical Reactivity with SH Compounds. J. Antibiot. 1975, 28, 743-748). In the 25 years since those findings were reported, the inhibition of GlxI by 2a has come to be associated with its antitumor activity, although no direct evidence supports such a causal relationship, and the hypothesis has never been tested.

Recently, it has been observed that the simpler COTC analog 1b (COMC) also displayed potent antitumor activity against a range of different murine and human tumors in culture, with 1b being somewhat more potent (IC50=0.5-19 μM) than 1a (IC50=3-44 μM) (Aghil, O.; Bibby, M. C.; Carrington, S. J.; Doubic, J.; Douglas, K. T.; Phillips, R. M.; Shing, T. K. M. Synthesis and Cytotoxicity of Shikimate Analogues. Structure: Activity Studies Based on 2-Crotonyloxymethyl-3R,4R,5R-trihydroxycyclohex-2-enone. Anti-Cancer Drug Design 1992, 7, 67-82). The endocyclic enone function in these compounds was required for antitumor activity. The antitumor activities of 1b were also thought to result from inhibition of GlxI by the corresponding GSH adduct 2b. Neither structure 2a nor 2b had been isolated or characterized, and the structures proposed for 2a and 2b were assigned indirectly, on the basis of model experiments with simple thiols.
GlxI plays a pivotal role in detoxifying intracellular methylglyoxal, which is formed during normal carbohydrate metabolism (Vander Jagt, D. L. The Glyoxalase System. In Coenzymes and Cofactors: Glutathione; Dolphin, D.; Poulson, R.; Avramovic, O., Eds.; John Wiley and Sons: New York, 1989; Vol. 3 (part A), pp 597-641.; Creighton, D. J. and Pourmotabbed, T. in Molecular Structure and Energetics: Principles of Enzyme Activity, Liebman, J. F. and Greenberg, A., Eds., VCH Publishers, New York 1988, Vol. 9. pp 353-386). Certain inhibitors of human GlxI, the most potent of which are enediol transition state analogue inhibitors, have been shown to retard the growth of both murine and human tumors in culture and in tumor-bearing mice by causing the accumulation of intracellular methylglyoxal (Kavarana, M. J.; Kovaleva, E. G.; Creighton, D. J.; Wollman, M. B.; Eiseman, J. L. J. Med. Chem. 1999, 42, 221-228; Sharkey, E. M.; O'Neill, H. B.; Kavarana, M. J.; Wang, H.; Creighton, D. J.; Sentz, D. L.; Eiseman, J. L. Cancer Chemother. and Pharmacol. 2000, 46, 156-166).
While a priori plausible, the hypothesis of Takeuchi et al. for the antitumor action of COTC and COMC has never been tested. Recently, authentic samples of both 2a and 2b were prepared by independent synthesis, and shown to be only modest inhibitors of GlxI (Huntley, C. F.; Hamilton, D. S.; Creighton, D. J.; Ganem, B. Org. Lett. 2000, 2, 3143-3144; Hamilton, D. S.; Ding, Z.; Ganem, B.; Creighton, D. J. Glutathionyl Transferase Catalyzed Addition of Glutathione to COMC: A New Hypothesis for Antitumor Activity. Org. Lett. accepted and in press) (FIG. 1).